Culture Media & Culture Methods PDF

Culture Media &Culture Methods Wijini Wijayabandara (BSc. Biomedical Science; MSc. Molecular Life Sciences)  Culture Media Bacteria have to be grown (cultured) for them to be identified and studied Should be grown separately (isolated) on a media and needed to be...

Culture Media &Culture Methods Wijini Wijayabandara (BSc. Biomedical Science; MSc. Molecular Life Sciences)  Culture Media Bacteria have to be grown (cultured) for them to be identified and studied Should be grown separately (isolated) on a media and needed to be obtained in the purest form A culture medium is essentially composed of basic elements (water, nutrients), to which growth factors that will be specific to each bacterium is added to facilitate their growth. History The first liquid artificial culture medium was created by Louis Pasteur in 1860 In 1881, Robert Koch demonstrated optimal growth of bacteria when they were incubated in a broth composed of fresh beef serum or meat extract. However, the use of a liquid culture medium did not produce pure bacterial cultures. Koch therefore sought a way to solidify the medium 2  Culture Media contd. Chemical substances can be added to culture media to inhibit certain bacteria. These inhibiting substances include potassium tellurite and bile salts, which inhibit Gram- positive bacteria or lithium chloride which eliminates Gram-negative bacteria One of the first gelling agents used in culture media was gelatin. The problem with this gelling agent is that it melts at 37°C, which is the incubation temperature of most bacteria Agar was then used in culture media. However, over-consumption of agar has led to a reduction in its source, red algae, which has increased costs. In addition, agarase, present in some bacteria, destroys agar, preventing the isolation of these bacteria Agar can cause inhibition of PCR of fungal DNA when extracted directly from the solid culture medium New gelling agents have been sought like sodium alginate, high-methoxyl and low-methoxyl pectins and gellan gum  Agar Agar, also called Agar-Agar, is a natural gelatinous substance, extracted from several species of red algae, mainly of the Gelidium and Gracilaria type No nutritive value Melts at 98°C & sets at 42°C 1-2% agar is employed in solid medium Smaller amounts (0.05-0.5%) are used in media for motility studies 4  Colony Bacteria grow on solid media as colonies A colony is defined as a visible mass of microorganisms all originating from a single mother cell Colony morphology is the visual culture characteristics of a bacterial colony on an agar plate Observing colony morphology is an important skill used in the microbiology laboratory to identify microorganisms 5 Colony contd. 6  Types of culture media 1. Based on their consistency 1. solid medium 2. liquid medium 3. semi solid medium 2. Based on the constituents/ ingredients 1. simple medium 2. complex medium 3. synthetic or defined medium 4. Special media 7  Types of culture media contd. Special media 1. Enrichment media 2. Selective media 3. Indicator media 4. Differential media 5. Transport media 6. Media for biochemical reactions Based on Oxygen requirement 1. Aerobic media 2. Anaerobic media 8  Classification based on consistency Solid media – contains 1-2% agar Solid culture media are obtained by adding a gelling agent, such as agar, to the culture broth. They make it possible to obtain isolated colonies of different bacterial species, which can be identified. Eg: Nutrient agar, Blood agar Liquid media – no agar. In liquid culture media, also called culture broths, nutrients are dissolved in water. The growth of bacteria in this type of medium can be demonstrated by the appearance of a turbidity in the medium this type of culture medium does not allow the morphological characteristics of bacterial species to be identified Eg: Nutrient broth Semi solid medium – 0.2-0.5% agar. Semi Solid Media have a soft jelly-like consistency which is used for the determination of bacterial motility. Eg: Hugh and Leifson's oxidation fermentation medium, Stuart's and Amies media, and Mannitol motility media. 9  Classification based on constituents Simple / basal media The simplest and most common media used in laboratories Aids in identification of Non-fastidious bacteria (ones which do not require any special nutrition supplement or conditions to grow) Ex. Escherichia coli, Staphylococcus aureus Eg: nutrient broth (NB), peptone water, and nutrient agar NB consists of peptone, meat extract, NaCl NB + 2% agar = Nutrient agar (NA) 10 Classification based on constituents Complex media Other than simple media, all forms of media are termed complex media Complex media contain additional components for bringing out specific qualities or providing the unique nutrients needed for the bacterium’s growth Eg: MacConkey agar medium, chocolate agar Synthetic or defined media A synthetic medium is a defined medium A defined medium (chemically defined medium) is a medium in which there is no yeast, plant or animal tissue present, and all the chemicals employed are known These media are very useful for studying the physiology, metabolic nature and nutritional requirements of bacteria Eg: Simmons citrate agar, mineral glucose medium 11 Special media –Enrichment media Enrichment media -solid Enriched media contain the nutrients required to support the growth of a wide variety of organisms Used to increase a small number of desired organisms to detectable levels Substances like blood, serum, egg are added to the basal medium Blood, which provides hemin and other nutrients. This component is frequently added to agar media to increase the growth of anaerobic bacterial species Eg: Blood agar, Chocolate agar Blood agar is usually prepared from tryptic soy agar or Columbia agar base with 5% sheep blood Ex. Streptococcus pneumoniae and Neisseria species Chocolate agar- Cornstarch, Potassium phosphate, Potassium phosphate, Sodium chloride Ex. Neisseria spp., and Gardnerella vaginalis Haemophilus influenzae 12  Special media –Enrichment media Enrichment media- liquid Liquid media used to isolate pathogens from a mixed culture Media is incorporated with inhibitory substances to suppress the unwanted organism Eg: Selenite F Broth – for the isolation of Salmonella, Shigella Alkaline Peptone Water – for Vibrio cholerae 13  Special media- Selective media Selective media A selective culture medium is used to isolate a particular bacterial species After the addition of a number of inhibitors to the culture medium, the objective of this type of medium is to eliminate unwanted microbial flora Eg: Mac Conkey’s medium for gram negative bacteria TCBS Agar (Thiosulfate-Citrate-Bile-Sucrose Agar) is a selective differential medium for isolating and cultivating Vibrio cholerae and other Vibrio species from clinical specimens Lowenstein-Jensen (LJ) medium – M.tuberculosis Potassium tellurite medium – Diphtheria bacilli Potassium Tellurite LJ 14 Special media - Indicator media It contains certain indicators like dyes or metabolic substrates in the medium composition which gives different colors to colonies of different microbial species when they utilize or react with these components Eg: Blood agar - In blood agar, three types of blood cell lysis or hemolysis are observed S. pyogenes completely lyse blood cells thus causing total clearing of the media around its colonies. E.coli partially lyse red blood cells, resulting in a greenish-colored medium, while microorganisms like Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus epidermidis, can’t lyse red blood cells, thus causing no color change in the medium Mac Conkey’s medium - It differentiates the gram-negative bacteria based on their lactose metabolism. The lactose fermenting bacteria, such as Escherichia coli, Klebsiella spp, Citrobacter, and Enterobacter forms pink-red colonies, while lactose non-fermenters, like Salmonella, Shigella, Proteus, Providencia, Pseudomonas, and Morganella form pale or colorless colonies 15  Special media - Differential media Differential media A media which has substances incorporated in it enabling it to distinguish between bacteria Eg: Mac Conkey’s medium Peptone Lactose Agar Neutral red Taurocholate Distinguish between lactose fermenters & non lactose fermenters. 16 Special media - media for biochemical reactions Sugar media Media containing any fermentable substance Eg: glucose, arabinose, lactose, starch etc. Media consists of 1% of the sugar in peptone water Contain a small tube (Durham’s tube) for the detection of gas by the bacter 17  Special media - Transport media Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent Buffered glycerol saline – enteric bacilli 18 Culturing of bacteria 19  Oxygen requirement Aerobic → obligate aerobe, require O2 to produce energy and growth Anaerobic → obligate anaerobe, do not require O2 , no growth if Oxygen is present Facultative → grow with or without O2, produce energy by fermentation/ anaerobic respiration in absence of O2 Microaerophilic → prefer reduced O2 tension (2-10%) 20  Based on the oxygen requirement Anaerobic media These media are used to grow anaerobic organisms. Eg: Robertson’s cooked meat medium 21  Biochemical Test & Reactions They provide additional information for the identification of the bacterium The tests include: Oxidase test Catalase test Indole test Citrate utilization Urease test 22  Oxidase Test Detects the presence of an enzyme “oxidase” produced by certain bacteria which will reduce the dye – tetramethyl-p-phenylene diamine dihydrochloride Positive test is indicated by the development of a purple colour Oxidase positive – Pseudomonas, Vibrio, Neisseriae Oxidase negative – Salmonella, Shigella 23  Catalase Test If the bacteria possess catalase (i.e., are catalase-positive), when a small amount of bacterial isolate is added to hydrogen peroxide, bubbles of oxygen are observed The catalase test is done by placing a drop of hydrogen peroxide on a microscope slide Morphologically similar Streptococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test 24  Indole Test Used to detect indole production by the organism. They produce indole from tryptophan present in peptone water After overnight incubation, a few drops of indole reagent (Kovac’s reagent) is added Positive test is indicated by a pink ring Positive indole test – pink ring Negative indole test - yellow ring Indole positive – E.coli Indole negative – Klebsiella, Salmonella. 25  Citrate Utilization Done in Simmon’s Citrate medium To detect the ability of certain bacteria to utilize citrate as the sole source of carbon Contains Sodium citrate and bromothymol blue as the indicator If citrate is utilized, alkali is produced which turns the medium to blue Citrate positive – blue colour Citrate negative – green colour Positive – Klebsiella Negative – E.coli 26  Urease Test Done in Christensen’s urease medium This test is used to detect organisms that produce urease Urease produced by the organisms split urea into ammonia and CO2 Urease positive – pink colour Urease negative – yellow colour Positive – Proteus, Klebsiella Negative – E.coli, Salmonella 27  Antibiotic Susceptibility Testing Susceptibility is a term used when microbe such as bacteria and fungi are unable to grow in the presence of one or more antimicrobial drugs Main two methods of ABST Disk diffusion method Dilution method 28  Disk diffusion Method q Disk diffusion by the Kirby-Bauer method is a standardized technique for testing rapidly growing pathogens q Fresh bacterial sub culture is swabbed onto the surface of Mueller–Hinton (MH) agar q Filter paper disks impregnated with a standardized concentration of an antimicrobial agent are placed on the surface, and the size of the zone of inhibition around the disk is measured after overnight incubation q Specific incubation time ranges are outlined in the Clinical and Laboratory Standards Institute [CLSI] documents Sensitive(s):Zone radius is wider than or equal to or not more than 3mm smaller than the control Intermediate(I): Zone radius is >2mm but smaller than the control by >3mm Resistant(R): No zone of inhibition or zone radius measures 2mm or less 29  Synergism And Antagonism Synergism, in general, may be Antagonism is the opposite of defined as two or more agents Synergism, i.e. two or more working together to produce a agents working in opposite result not obtainable by any of the actions agents independently. 30  Dilution Method q The Dilution method is used to determine the minimal inhibitory concentration of an antimicrobial to inhibit or kill the bacteria/fungi q MIC is the lowest concentration of an antimicrobial agent (in mg/mL), which prevents the appearance of visible growth of a microorganism q Initially, serial dilutions of the antimicrobial agent are prepared q Each tube is inoculated with a known number of test microorganisms q After a suitable incubation period, bacterial growth is assessed by bacteria colony counting/ turbidity of suspension and an MIC is determined In microbiology, McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing. An example of such testing is antibiotic susceptibility testing by measurement of minimum inhibitory concentration which is routinely used in medical microbiology and research 31 McFarland solution 32 THANK YOU Affiliated with Durdans Hospital 33